Authors: Caroline Cowley, Megan Jewell, Brenda Mendoza, Aubrey Cluff, Ryan Summerhays, Jason Hansen
Mentors: Jason Hansen
Insitution: Brigham Young University
Approximately 8 million newborns manifest a birth defect every year worldwide. One of the most common birth defects involve disruptions in musculoskeletal development. Oxidative stress has been found to propagate teratogenesis. Thioredoxin-1 (Trx1), an oxidoreductase, is an important antioxidant regulator required for proper embryonic development. Trx1 knockouts have been found to be embryolethal prior to implantation. A preliminary study to assess osteogenesis was conducted using mouse embryonic fibroblasts (MEFs) originating from transgenic conditional Trx1 knockout embryos. Upon confluence, MEFs were stimulated to undergo osteogenesis via commercially available media. A subset of cells were treated with doxycycline (DOX) prior to and throughout the culture period. MEFs were maintained over a 21 day period in a reduced oxygen environment. MEFs were then fixed in formalin and stained with Alizarin red to determine the degree of osteogenesis. MEFs treated with DOX were unable to undergo proper osteogenesis. While this would suggest that osteogenesis is regulated through proper functions of Trx1, it is unknown how Trx1 regulates osteogenesis in utero. Because Trx1 deletion is lethal prior to implantation it has been historically difficult to study the role of Trx1 during organogenesis. With the development of the DOX-inducible Trx1 conditional knockout mouse, we can now target specific developmental periods and evaluate post-implantation processes like osteogenesis. Using proper transgenic mice and breeding schemes, DOX-inducible Trx1 conditional knockout embryos were treated in utero with DOX through the dam’s drinking water, starting on gestational day (GD) 8.5. The embryos were collected on GD 16.5, fixed in 95% ethanol, and then skinned. To visualize bone and cartilage, the embryos were placed in ethanol and subsequently stained with Alizarin red and Alcian blue. The staining showed that embryos lacking Trx1 were significantly stunted in their skeletal maturation. With this data, we are the first to show that during organogenesis, the musculoskeletal system is affected by deletions of Trx1 at specific periods of development. Under oxidizing conditions which exceed the capacity of the oxidoreductase pathway of Trx1, Trx1 exists primarily in its oxidized form and can no longer reduce proteins that have been turned off by oxidation. Our Trx1 deletions model a highly oxidized state in which Trx1 is dysfunctional. Because regulatory redox control of protein activity is required for proper embryonic development, exposure to oxidizing environmental conditions specifically affecting Trx1 redox state may target the disruption of the musculoskeletal system.